In vitro*HQHUDWLRQRI*HUP&HOOV7VJDDVD0RGHO

Original Article Evaluation of in vitro Spermatogenesis System Effectiveness to Study Behavior: Monitoring the Expression of the Testis 6SHFL¿F 7VJD *HQHDVD0RGHO

Mohammad Miryounesi MD PhD1,2, Karim Nayernia PhD3, Maryam Beigom Mobasheri MSc2,4, Mahdi Dianatpour PhD5, Richard Oko PhD6, Shahram Savad MD PhD7, Mohammad Hossein Modarressi MD PhD‡2

Abstract Background: In vitro generation of germ cells introduces a novel approach to male infertility and provides an effective system in tracking studies, however many aspects of this process have remained unclear. We aimed to promote mouse embryonic stem cells (P(6&) differentiation into germ cells and evaluate its effectiveness with tracking the expression of the Tsga10 during this process. Methods: P(6&V were differentiated into germ cells in the presence of Retinoic Acid. Based on developmental schedule of the postnatal testis, samples were taken on the 7th, 12th, and 25th days of the culture and were subjected to expression analysis of a panel of germ cell VSHFL¿F genes. ([SUHVVLRQ of Tsga10 in RNA and levels was then analyzed. Results: Transition from mitosis to meiosis occurred between 7th and 12th days of P(6& culture and post-meiotic gene expression did not occur until the 25th day of the culture. Results showed low level of Tsga10expression in undifferentiated stem cells. During transition from meiotic to post-meiotic phase, Tsga10 expression increased in 6.6 folds. This ¿QGLQJ is in concordance with in vivo changes during transition from pre-pubertal to pubertal stage. Localization of processed and unprocessed forms of the related protein was similar to those in vivo as well. Conclusions: ([SUHVVLRQ pattern of Tsga10, as a gene with critical function in spermatogenesis, is similar during in vitro and in vivo germ cell generation. The results suggest that in vitro derived germ cells could be a trusted model to study genes behavior during spermatogen- esis.

Keywords: Tsga10; embryonic stem cells; differentiation; in vitro germ cells generation

Cite this article as: Miryounesi M, Nayernia K, Mobasheri MB, Dianatpour M, Oko R, Savad S, Modarressi MH. Evaluation of in vitro Spermatogenesis System Effectiveness to Study Genes Behavior: Monitoring the Expression of the Testis Specific 10 (Tsga10) Gene as a Model. Arch Iran Med. 2014; 17(10): 692 – 697.

Introduction Consequently we tried to create an in vitro spermatogenesis sys- tem and monitor a testis VSHFL¿F gene, Tsga10, in the process of bout 15% of couples have fertility problems and male fac- stem cells differentiation, and compare it with in vivo system. This tor infertility accounts for half of the cases.1 Numerous gene has been studied in our team in several research projects and A pathologic conditions are involved in infertility prob- adequate information about its in vivo characteristics has been ob- lems.2 To elucidate the causes of male infertility, it is important to tained. know about the mechanisms of germ cell VSHFL¿FDWLRQ, develop- The Tsga10 is mainly expressed in the testis tissue. It has been ment and differentiation. Due to lack of a robust in vitro culture shown that Tsga10 has a critical role in spermatogenesis and its system for differentiation of germ line progenitor cells into mature protein contributes to the ¿EURXV sheath of sperm tail.5 In addition gametes, enough knowledge in this area is not available. Tsga10 is expressed in actively dividing cells and fetal tissues.6 Recent studies have shown that embryonic stem cells (ESC) can This gene is a member of cancer testis (CT)7,8 genes family. Its give rise to primordial germ cells (PGCs) and sperm-like cells in aberrant expression is reported in acute lymphoblastic leukemia vitro.3 This ability provides an in vitro model to analyze the biol- and some primary cancers.9 Tsga10 mRNA is translated into a ogy of germ cells development and new therapeutic approaches in 65-kilodaltone protein. During spermatogenesis, it is processed to reproductive medicine.4 a 27-kilodalton N-terminal segment. This portion is localized to the ¿EURXV sheath of mature spermatozoa.10 $XWKRUV¶DI¿OLDWLRQV 1Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2Department of Medical Genetics, School of In this study, for the ¿UVW time the alterations in Tsga10 expres- Medicine, Tehran university of Medical Sciences, Tehran, Iran. 3GENEOCELL, sion during in vitro differentiation of embryonic stem cells to 4 Advanced Molecular and Cellular Technologies, Montreal, Canada. Cancer Re- germ cells was analyzed. search Center, Tehran University of Medical Sciences, Tehran, Iran. 5Department of Medical Genetics, School of Medicine, Shiraz university of Medical Sciences, To FRQ¿UP the different developmental stages of the differenti- Shiraz, Iran. 6Departments of Anatomy and Cell Biology, Queen’s University, 9th ated cells, the expression panel of 6 stage VSHFL¿F genes, Stra8, 7 Floor, Botterell Hall, Kingston, Ontario, Canada K7L 3N6. Kamali Hospital, Sycp3, Dazl, Prm1, Spata19, and Plcz in the development process Alborz University of Medical Sciences, Karaj, Iran. ‡&RUUHVSRQGLQJ DXWKRU DQG UHSULQWV Mohammad Hossein Modarressi MD was studied. PhD, Department of Medical Genetics, School of Medicine, Tehran University Retinoic Acid (RA) is now FRQ¿UPHG to be a key signaling mol- of Medical Sciences, Tehran, Iran. Telefax: +98-218-895-3005, ecule in meiosis induction.11 RA induces a VLJQL¿FDQW increase in E-mail: [email protected] Accepted for publication: 16 July 2014 the expression of Stra8 (a pre-meiotic gene) and Sycp3 (a meiotic

692 Archives of Iranian Medicine, Volume 17, Number 10, October 2014 00LU\RXQHVL.1D\HUQLD0%0REDVKHULHWDO gene) in cultures of fetal testis.12 According to gene knock- for two weeks. Resulting colonies were cultured for 25 days on out studies, Stra8 is believed to be the gatekeeper of the mitotic/ gelatin with RA treatment (¿QDO concentration 10-8 mol). meiotic switch.13 Dazl is a testis-VSHFL¿F gene with a critical role in spermatogenesis VSHFL¿FDOO\ in meiosis initiation.14,15 Sycp3 is a RNA extraction, cDNA synthesis and RT-PCR meiotic VSHFL¿F gene responsible for the formation of the synap- Total RNA was extracted using TRIzol reagent (Invitrogen) ac- tonemal complex. This complex has an important role in synapse cording to the manufacturer’s protocol. RNA concentration was formation, recombination and segregation of dur- measured with Nano Drop 1000 spectrophotometer (Thermo ing meiosis.16 Sycp3 is positively regulated by Dazl through bind- Fisher 6FLHQWL¿F). The extracted RNA (2 μg) was used for cDNA ing to 3ƍUTR of its transcript. Protamine is coded by Prm1 which synthesis using M-MuLV reverse transcriptase (Fermentase) with is a post-meiotic gene with VSHFL¿F expression in haploid round random hexamer and oligo dT primer together. RT-PCR was per- spermatids. During development of mature spermatozoa histones formed using VSHFL¿F primer for EGFP, Oct4, Stra8, Sycp3, Dazl, are replaced by protamines.17,18 Spata19 is a testis VSHFL¿F gene Prm1, Plcz, and Spata19 genes (Table 1). that is expressed VSHFL¿FDOO\ in round spermatids.19 Finally, Plcz is the physiological agent of oocyte activationand its protein causes Real time RT-PCR Ca2+ osciliation in oocyte similar to those seen in fertilization. It Real time RT-PCR was carried out using VSHFL¿F primer for was shown that Plcz mRNA is ¿UVW detectable in spermatids.20 Tsga10 gene with StepOne Real time PCR system (Applied Biosystems). Typically, a 20 μL reaction contained 10μL 2 × Materials and Methods SYBR® Green PCR Master Mix (Applied Biosystems), 1 μL (¿- nal 500nM) forward and reverse primer, 1 μL of cDNA and 8 μL Cell culture distilled water. Expression analysis of Tsga10 gene and a house- Mouse embryonic stem cell line C57BL6 with normal male keeping gene, Tbp (Tata Binding Protein) were performed using (XY) karyotype (Invitrogen) was cultured in an undifferenti- StepOne Software v2.1 (Applied Biosystems). ated state on a feeder layer of mitomycin-C inactivated mouse embryonic ¿EUREODVW.21,22 Culture medium composed of Knock- Western blot analysis out Dulbecco’s PRGL¿HG Eagle’s medium (Knockout DMEM, Equal amounts of cell lysate of selected samples (adult GIBCO-BRL) supplemented with 12.5% (v/v) ES TXDOL¿HG FBS testis and differentiated ES) were separated using 10% SDS- (GIBCO-BRL), 2mmol L-Glutamine (GIBCO-BRL), 1X nones- PAGE and transferred to a Polyvinylidene ÀXRULGH (PVDF) mem- sential amino acids (NEAA; GIBCO-BRL), 50 μgml-1 penicillin brane (Milipore). The membrane free binding sites were blocked and streptomycin, as well as 50 μmol ȕ-mercaptoethanol and 10³ by treating the membrane with 5% (wt/ vol) skim milk for one unit ml-1 LIF (Milipore). hour at room temperature. After overnight incubation at 4°C with primary Tsga10 goat polyclonal antibody (Santa Cruz), blots were &RQVWUXFWLRQ RI JHUP FHOO VSHFL¿F UHSRUWHU JHQH DQG UHFRPELQDQW washed 3 times with PBST. After incubation with the appropri- mESCs ate horseradish peroxidase-labeled secondary antibody (anti goat, According to our previous published data, a segment of Stra8 Santa Cruz), blots were washed again as mentioned previously. gene promoter (-1400/+7) was DPSOL¿HG from genomic DNA Bands were detected using enhanced chemiluminescence (ECL). and inserted in the SacI/HindIII site of PRGL¿HG pEGFP-1 vector where neomycin-resistance cassette was replaced with puromy- Immunohistochemistry of TSGA10 in seminiferous tubules cin-resistance gene (Figure 2d).23 ES cells were trypsinized and Sections from testes (5-ȝP) ¿[HG in Bouin’s and embedded in paraf- approximately 7 × 106 cells were prepared for performing elec- ¿Q, were GHSDUDI¿QL]HG in xylene and hydrated through a graded se- troporation. Linearized vector (35 μg) was electroporated into ries of ethanol solutions. During hydration the sections were treated to ES cells. Electroporation was performed on Bio-Rad GenePulser abolish the endogenous peroxidase activity, neutralize residual picric device at 250 V and 500 μF. After electroporation, mESCs were acid and block free aldehyde groups. Once hydrated, the sections were transferred to a 6-well plate and cultured 72 hours without antibi- blocked with 10% goat serum in TBS for 15 minutes, incubated with otic. Then puromycin (¿QDO concentration 1 μg ml-1) was added to primary antibodies (polyclonal anti whole TSGA10 protein and poly- medium and cells were cultured in the presence of puromycin for clonal anti N-terminal) overnight at 4ºC, or at 21ºC for 2 hr, washed in three weeks. Puromycin resistant colonies were selected. DNA TBS containing 0.1% Tween-20 (4 × 5 min.), blocked in 10% NGS from these colonies was extracted and tested for the presence of in TBS and incubated with anti-goat IgG conjugated to peroxidase Stra8/EGFP vector by PCR and positive colonies were grown in (Santa Cruz) in TBS (1:250) for 1 hr. After an extensive washing in complete medium with LIF for one month.24 TBS containing 0.1% Tween-20, peroxidase reactivity of the sections was tested by incubating the sections with 0.03% hydrogen peroxide Derivation of germ cells from mESCs and 0.05% diaminobenzidene tetrahydrochloride (DAB) in TBS con- RA (Sigma) was added to the medium at a ¿QDO concentration of taining 0.1 M imidazole, pH 7.6 for 10 min. The sections were then Ø5 mol for 72 hours to induce differentiation. GFP-positive cells washed with double distilled water, counterstained with 0.1% ¿OWHUHG were selected using ÀXRUHVFHQFH-activated cell sorting (FACS). methylene blue, and immersed in tap water for 5 min. Following stain Cells were trypsinized and after pipetting up and down several differentiation the sections were dehydrated, cleared in xylene and times ¿OWHUHG through a 40 ȝP strainer (Falcon) to create single- mounted with coverslips using Permount.25 cell suspensions. Sorting was carried out with 3×106 cells’ using BD FACS Aria II ÀRZ cytometer and undifferentiated ESCs were Results used as a negative control.23 These cells were cultured on a mouse embryonic ¿EUREODVWV (MEF) feeder under non-induced condition Establishment of spermatogonial stem cell (SSC)-like cells

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Table 1. Primer sequence and expected size of PCR products of the Oct4, Stra8, EGFP, Dazl, Sycp3, Spata19, prm1, Plcz, and Tsga10 genes.

Gene Primer Sequence Product size (bp)

F: CTGAAGCAGAAGHAGGATCACC Oct4 345 R: TCGAACCACATCCTTCTCTAGCC F : ACAACCTAAGGAAGGCAGTTTAC Stra8 174 R: TGACCTCCTCTAAGCTGTTGG F: GCACCATCTTCTTCAAGGACGAC EGFP 270 R: TCTTTGCTCAGGGCGGACTG F: CAGGCATATCCTCCTTATCCAAG Dazl 263 R: TGTATGCTTCGGTCCACAGAC F: CCGGAGCCGCTGAGCAAACA Sycp3 430 R: CCAGTTCCCACTGCTGCAACAC F: CTCACAGGTTGGCTGGCTCGAC Prm1 192 R: CGGCGACGGCAGCATCTTCG F: CTGAAGATGATCATTACA Spata19 475 R: TTAGCATTCTGAGCAAGAAGTC F: TGGCCTTATCTGATCTTGT Plcz 256 R: GCCACCATCTGACAACCTA F: AGACCTTCCTCCTTTATGAGCA Tsga10 141 R: AAGGCTACATCTCTCTCCGTC

Table 2.([SUHVVLRQSDWWHUQRIVWDJHVSHFL¿FJHQHVGXULQJGLIIHUHQWLDWLRQRIP(6&VWRJHUPFHOOV

Nanog Oct4 Stra8 Dazl Sycp3 Prm1 Spata19 Plcz Un differentiated mESCs + + íííí í í After 72 h induction with RA + + + ííí í í Day 7 + + + + íí í í Day 12 + + + + + íí í Day 25 + + + + + + + +

Figure 1. a) RT-PCR analysis of saples (7th ,12th, and 25th days) for meiot- Figure 2. a)'LDJUDPRI*)3H[SUHVVHGP(6&VVRUWHGE\)$&6b and ic (Dazl and Sycp3) and post-meiotic (Prm1 and Plcz) genes. b)([WUDFWHG c) $IWHUFXOWXUHRQ0()IHHGHUVRUWHGFHOOVVKRZHGKLJKH[SUHVVLRQRI RNA from samples were analyzed by gel electrophoresis *)3d) 0RGL¿HGS(*)3YHFWRUZKLFKStra8 promoter was inserted in multiple cloning sites

In order to isolate differentiated stem cells, a VSHFL¿F reporter cells were shown to be GFP-positive by FACS (Figure 2a). These construct consisting of a germ line-VSHFL¿F segment of Stra8 gene sorted cells were cultured under normal conditions (complete me- promoter and the coding region of enhanced green ÀXRUHVFHQFH dium plus LIF) for two weeks to increase cell viability (Figures protein (EGFP) were used. The use of this 1.4 Kb Stra8 promoter 2b and 2c). Differentiated cells were subjected to RNA extraction resulted in the testis-VSHFL¿F expression of GFP. After selection (Figure 1b), cDNA synthesis and analyzed for the expression of with puromycin, ES colonies were picked up and PCR with GFP the six germ cell VSHFL¿F genes. RT-PCRs were performed for VSHFL¿F primers was performed to verify the presence of the con- Stra8 and Oct4 as pre-meiotic genes, Dazl and Sycp3 as meiotic struct in the genomic DNA of each colony. Induction with RA genes and Prm1 and Spata19 as post-meiotic genes. Oct4 was ex- (¿QDO concentration of 10-5 mol) was performed for 72 hours. GFP pressed in both induced and non-induced ES cells. Stra8 showed expression was detected after RA treatment and colonies that were high expression after 72 hours induction with RA. Expression of not induced with RA had no GFP shine. Approximately 60% of Dazl, Sycp3, Prm1, and Spata19 was not detected in induced and

694 Archives of Iranian Medicine, Volume 17, Number 10, October 2014 00LU\RXQHVL.1D\HUQLD0%0REDVKHULHWDO

Figure 3. Real time RT-PCR results show elevation of Tsga10 expression in post meiotic germ cells. A)XQGLIIHUHQWLDWHGP(6&V B) +12 day sample, C) +25 day sample, D)P\RFLWHGULYHGIURPP(6&E) 15-day-old mouse testis, F) adult mouse testis

Figure 4. Analysis of TSGA10 protein, a) Western blot analysis showed a prominent 65-kilodalton and 27-kilodalton band in sample of adult Mouse testis. b)7KHVHEDQGVLQ3RVWPHLRWLFJHUPFHOOVGULYHGIURPP(6&. C) NLORGDOWRQEDQGLQXQGLIIHUHQWLDWHGP(6& d),PPXQRKLVWRFKHPLVWU\RI6HPLQLIHURXVHSLWKHOLXP 6( LQVWDJH,,,,9RIWKHF\FOHRIWKH6(VKRZLQJLQWHQVHLPPXQRVWDLQLQJLQ the cytoplasm of round spermatids (arrow) and more moderate immunostaining in pachytene spermatocytes. Note the immunreac- tive tails of elongated spermatids in the lumen. e) 6(LQVWDJH9,,,PPXQRVWDLQLQJLVIRXQGLQWKHF\WRSODVPRIHORQJDWHGVSHUPDWLGV (arrow) ready to be extruded into the lumen. Note the cytoplasmic droplet and principal piece of the tail are immunoreactive.

non-induced cells. Stra8 is a molecular marker for spermatogonial a single peak and agarose gel electrophoresis FRQ¿UPHG the re- stem cells26 so it was revealed that induced cells were spermatogo- sults. Expression SUR¿OH of Tsga10 in selected samples on 12th nial stem cell (SSC)-like cells. and 25th days after initiation of RA treatment was investigated. Undifferentiated mESC showed low expression of Tsga10 which Differentiation of SSC-like cells to germ cells was assigned to 1 for data analysis by StepOne Software. Results SSC-like cells were cultured in medium supplemented with RA showed that during early phase of mESC differentiation to germ (10-8 mol) and mRNA of these cells was extracted on 7th, 12th, cells (12th day), relative Tsga10 expression value dropped to 0.32. and 25th days after initiation of treatment with RA. These samples The relative expression of Tsga10 was increased 2.2 fold in con- were analyzed for pre-meiotic (Oct4 and Stra8), meiotic (Sycp3 trast to undifferentiated mESC after expression of post meiotic and Dazl), and post-meiotic (Prm1 and Spata19) genes expres- genes (25th day). As a control the expression of Tsga10 decreased sion. On the 7th day of cell culture Oct4, Stra8, and Dazl genes to 0.12 in the mESC differentiated into myocyte (details of the were expressed, but expression of Sycp3 was initiated on the 12th differentiation method not shown). In vivo analysis showed that day of culture. Expression of Prm1, Plcz, and Spata19 did not the relative expression of Tsga10 in 15-day-old mouse testis was start until the 25th day of culture (Figure 1a). 140. In adult mouse the relative expression of Tsga10 was 1650 (Figure 3). Tsga10 was up-regulated during production of germ cells In western blot analysis, polyclonal TSGA10 antibody was Expression analysis of Tsga10 was performed by Real-Time used to identify the presence of TSGA10 protein. A prominent RT-PCR. The melting-curves of Tsga10 and Tbp products showed 65-kilodalton and a pale 27-kilodalton band for TSGA10 protein

Archives of Iranian Medicine, Volume 17, Number 10, October 2014 695 ,QYLWUR*HQHUDWLRQRI*HUP&HOOV7VJDDVD0RGHO were detected in the testis sample (Figure 4a). We detected the vivo studies showed up-regulation of Tsga10 expression, the value same bands in the post-meiotic germ cells derived from mESC of up-regulation was different in them, which may be due to dif- (Figure 4b) and a pale 65-kilodalton band in undifferentiated ferences in the HI¿FLHQF\ of differentiation in testis vs. monolayer mESC (Figure 4c). To examine localization of TSGA10 protein cell culture. in adult mouse testis, we performed immunohistochemistry. Only Reproductive biologists have limited available tools to deter- seminiferous tubules were immunostained. The results showed mine the function of genes involve in germ cell development. that full length TSGA10 (Figure 4d) is intensely expressed in These methods are laborious and time consuming.29 Based on our round spermatids (or early in spermiogenesis) and some cells ¿QGLQJ we have suggested that in vitro spermatogenesis could be appeared to be the early pachytene spermatocytes. The N-ter- an alternative method for germ cell development studies. minal portion of TSGA10 (Figure 4e) is strongly expressed in One of the best methods for analyzing the effect of genes in de- elongated spermatids, which are ready to detach from the semi- velopment is generating knockout mice. Building knockout mice niferous epithelium. is an expensive and time consuming method. In addition, in the ¿HOG of spermatogenesis, producing knockout mice is more dif- Discussion ¿FXOW because these knockout animals are usually infertile. An alternative method for knockout animal technology is producing In vitro differentiation of ESC provides a model for analyzing of knockout ES cells, in which single or both copies of a testis spe- developmental process. In the present study, we have developed FL¿F gene is deleted, and differentiated these cells toward germ a step-by-step differentiation model for analyzing stage VSHFL¿F cells. According to the similarity of in vitro and in vivo spermato- gene expression. genesis, the effect of the knockout gene in gametogenesis could During the transition of mammalian germ cells from mitosis to be analyzed. meiosis, Stra8 gene is expressed.25 A vector composed of promot- In conclusion, we suggest in vitro derived germ cells as appro- er region of Stra8 gene, and GFP coding sequence has been used priate models for analysis of germ cell VSHFL¿F genes expression. in this study to select stable transfected ES cells that entered meio- sis. We observed that if we did not separate GFP-positive cells, &RQÀLFWRILQWHUHVW which entered the meiosis stage in the cell culture, differentiation process could be suppressed by neighboring undifferentiated cell The authors declare that they have no FRQÀLFW of interest. populations. This ¿QGLQJ has also been observed previously17 and that is the reason for separation of cells using FACS. Acknowledgments In the mouse testis, the postnatal spermatogenesis development can be divided into three phases: proliferation of SSCs (days This study has been funded by TUMS Grant number 90-01-30- 1–7), initiation of meiosis and differentiation of SSCs into sper- 11868. We would like to thank Dr. Arshia Seddigh for his helpful matocytes, and ¿QDOO\ the haploid round spermatid generation comments. (days 8–20), spermiogenesis and production of elongated sper- matozoa (days 21–36).28 References The start of Sycp3 gene expression on the 12th day showed that after this period of time, SSC-like cells have been entered the mei- 1. Massart A, Lissens W, Tournaye H, Stouffs K. Genetic causes of sper- osis. Expression of Prm1 and Spata19 genes in 25th day of culture matogenic failure. Asian J Androl. 2012; 14: 40 – 48. FRQ¿UPHG that these cells are post-meiotic germ cells. 2. Van Golde RJ, Tuerlings JH, Kremer JA, Braat DD, Schoute F, Hoef- sloot LH. DAZLA: an important candidate gene in male subfertility. J Previous studies showed strong correlation between Tsga10 ex- Assist Reprod Genet. 2001; 18: 395 – 399. pression and spermatogenesis.5 Full length TSGA10 protein is 3. Silva C, Wood JR, Salvador L, Zhang Z, Kostetskii I, Williams CJ, 65-kilodalton and its 27-kilodalton N-terminal fragment is a major et al. 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